Project Summary/Abstract Over 40% of human cancers are driven by hyperactivated RTK/RAS/RAF/MEK/ERK signaling (MAPK pathway). Targeting MAPK signaling using small-molecule RAF or MEK inhibitors is a validated therapeutic strategy in cancer, but the antitumor activity of these drugs is commonly attenuated by various mechanisms of adaptive resistance. One such common mechanism is the result of relief of negative feedback which promotes upregulation of expression and activity of multiple Receptor Tyrosine Kinases (RTKs), which in turn activate RAS and downstream MAPK signaling in the presence of inhibitor. Further, questions relating with how MAPK- directed therapies can achieve a higher therapeutic index by minimally affecting normal tissue and how can they be optimally combined with immune checkpoint therapies remain largely unresolved. SHP2 (PTPN11) is a non-receptor protein tyrosine phosphatase that mediates signal transduction downstream of multiple RTKs by associating with GRB2 and other adaptor proteins to form a complex that promotes RAS activation. SHP2 has also been suggested to have an immunosuppressive role, but this function of SHP2 has also been relatively understudied. The recent development of potent and selective allosteric small-molecule inhibitors targeting SHP2 provided the opportunity to potentially overcome adaptive resistance by co-targeting both oncogenic signaling and feedback-induced RTK-mediated RAS activation in tumors dependent on deregulated MAPK signaling. Using one such SHP2 inhibitor, SHP099, we found that combinatorial targeting of SHP2 and MAPK signaling prevented adaptive resistance in defined subsets of MAPK-dependent tumors. In each MAPK-driven tumor analyzed, induction of p(Y542)SHP2, a surrogate marker of SHP2 activation, in response to MAPK inhibition was required for combined treatment sensitivity. The strategy was broadly effective in tumor models representing aggressive cancer types for which there are no targeted therapeutic options currently available, including Triple Negative Breast Cancer (TNBC) models, as well as tumors with RAS mutations at G12. In contrast, RAS(G13D)/(Q61X) mutations were associated with tumor resistance to the combination, revealing a hitherto unappreciated complexity of mutant-RAS signaling and variability in the dependence of different RAS mutants on upstream RTK/SHP2 signaling. Finally, using an in vitro co-culture tumor cells/T cells system we found that SHP2 inhibition enhances T cell function. Based on these observations, we now plan to use specific inhibitors and biochemical and cell-based methods to comprehensively study mechanisms that regulate wild- type and mutant RAS activity downstream of RTK/SHP2 signaling. We will further investigate ex vivo and in vivo for molecular and tumor type-specific determinants of response to combined SHP2 and MAPK inhibition, that may be used as potential biomarkers and of the effects of these therapies on normal tissue and the immune system. The goal is to use the mechanistic knowledge gained by these studies to develop novel effective combinatorial pharmacologic strategies for MAPK-driven cancers.